1. Field of the Invention
This invention relates to endoproteases, particularly a novel endoprotease termed furin endoprotease. The invention also relates to inhibitors of furin endoprotease activity. In particular, the invention relates to novel variants of .alpha..sub.1 -antitrypsin that specifically inhibit furin endoprotease activity. The invention also provides methods for using such inhibitors to attenuate or prevent biological proteolytic maturation of bioactive proteins and peptides in vivo and in vitro, in particular viral proteins and bacterial toxins. Therapeutic methods and pharmaceutical compositions of such inhibitors are also provided directed towards the alleviation and treatment of disease having microbiological etiology.
2. Information Disclosure Statement
Most biologically active peptides and proteins are synthesized initially as larger, inactive precursor proteins that are endoproteolytically cleaved during transit through the secretory pathway in the Golgi apparatus in cells expressing such proteins (see Barr, 1991, Cell 66: 1-3 for review). This system comprises an important common mechanism required for synthesis of biologically active proteins and peptides in yeast (Fuller et al., 1988, Ann. Rev. Physiol. 50: 345-362), invertebrates (Scheller et al., 1983, Cell 32: 7-22) and mammalian cells (Sossin et al., 1989, Neuron 2: 1407-1417). Examples of proteins produced in vivo by exocytotic transport through the Golgi are precursors of peptide hormones, neuropeptides, growth factors, coagulation factors, serum albumin, cell surface receptors, and adhesion molecules.
Morrison et al., 1985, J. Virol. 53: 851-857 disclose that F protein of Newcastle disease virus is processed through the exocytotic transport pathway in infected cells.
Perez & Hunter, 1987, J. Virol. 61: 1609-1614 disclose that the Rous sarcoma virus (RSV) glycoprotein is processed through the exocytotic transport pathway in infected cells.
Yamada et al., 1988, Virology 165: 268-273 disclose that F protein of mumps virus is processed through the exocytotic transport pathway in infected cells.
Randolph et al., 1990, Virology 174: 450-458 disclose that the prM protein of flaviviruses is processed through the exocytotic transport pathway in infected cells.
A common structural feature of molecules processed through the exocytotic transport pathway is the presence of basic residues or pairs of basic residues at the proteolytic processing site in the molecule. Examples include serum factors (Factor IX; Bentley et al., 1986, Cell 45: 343-348; proalbumin; Knowles et al., 1980, Science 209: 497-499; pro-von Willibrand factor; Bonthron et al., 1986, Nature 324: 270-273), viral polyproteins (human immunodeficiency virus (HIV) gp160; McCune et al., 1988, Cell 53: 55-67; RSV envelope protein; Perez & Hunter, 1987, J. Virol. 61: 1609-1614; yellow fever virus protein; Rice et al., 1985, Science 229: 726-733; measles virus protein; Richardson et at., 1986, Virology 155: 508-523; mumps virus protein; Waxham et al., 1987, Virology 159: 381-389; human cytomegalovirus protein; Spaete et al., 1990, J. Virol. 64: 2922-2931; varicella zoster virus protein; Keller et al., 1986, Virology 152: 181-191), growth factors (pre-protransforming growth factor .beta.; Gentry et al., 1988, Molec. Cell. Biol. 8: 4162-4168; epidermal growth factor; Gray et al., 1983, Nature 303: 722-725; pro-.beta.-nerve growth factor (NGF); Edwards et al., 1988, Molec. Cell Biol. 8: 2456-2464), receptors (insulin receptor; Yoshimasa et al., 1988, Science 240: 784-787); and bacterial toxins (see Stephen & Pietrowski, 1986, Bacterial Toxins, 2d ed. (Amer. Soc. Microbiol. Washington, D.C.) for review; anthrax toxin; Singh et at., 1989, J. Biol. Chem. 264: 11099-11102). The proteolytic processing site has been identified in some of these molecules.
Berger & Shooter, 1977, Proc. Natl. Acad. Sci. USA 74:3647-3651 disclose the sequence -RSKR- (SEQ ID No.:1) at the proteolytic processing site of pro-.beta.-NGF.
Bentley et al., 1986, ibid. disclose the sequence -RPKR- (SEQ ID No.:2) at the proteolytic processing site of the blood coagulation factor protein Factor IX.
McCune et al., 1988, ibid., disclose the sequence -REKR- (SEQ ID No.:3) at the proteolytic processing site of HIV gp160.
Ciepak et al., 1988, Biochem. Biophys. Res. Comm. 157:747-754 disclose the sequence -RVRR- (SEQ ID No.:4) at the proteolytic processing site of diphtheria toxin.
Vey et al., 1992, Virology 188: 408-413 disclose the sequence -RX(R/K)R- (SEQ ID No.:5) at the proteolytic processing site of influenza hemagglutinin.
Ogata et al., 1990, J. Biol. Chem. 265: 20678-20685 disclose the sequence -RSKR- (SEQ ID No.:1) at the proteolytic processing site of Pseudomonas exotoxin A.
Klimpel et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10277-10281 disclose the sequence -RX(R/K)R- (SEQ ID No.:5) at the proteolytic processing site of anthrax protective antigen.
Recently, an endoprotease termed furin has been isolated that specifically recognizes the recognition sequence of proteins processed through the exocytotic secretory pathway (Wise et al., 1990, Proc. Natl. Acad. Sci. USA 87: 9378-9382; Bresnahan et al., 1990, J. Cell Biol. 111: 2851-2859). This endoprotease is a subtilisin related, calcium-dependent serine protease (Bresnahan et al., ibid.). A complementary DNA copy of the mRNA encoding this endoprotease has been isolated (Wise et al., ibid.) and sequenced (van den Ouweland et al., 1992, Nucleic Acids Res. 18: 664) and expressed in heterologous cells (Bresnahan et al., ibid.). These studies have shown furin to be expressed as a doublet of 96 and 90 kilodaltons (kD) in size, ubiquitously expressed as a 4.5 kilobase (kb) mRNA, and localized by fluorescence immunohistochemistry to the Golgi apparatus of cells expressing this endoprotease (Bresnahan et al., ibid.). Furin has been shown to be capable of proteolytically cleaving a number of exocytotically processed proteins.
Bresnahan et al., ibid., disclose furin-mediated cleavage of pro-.beta.-NGF.
Wise et al., ibid., disclose furin-mediated cleavage of pro-von Willibrand factor and complement factor C3.
Hosaka et al., 1991, J. Biol. Chem. 266: 12127-12130 disclose furin-mediated cleavage of renin.
Steineke-Grober et al., 1992, EMBO J. 11: 2407-2414 disclose furin-mediated cleavage of influenza hemagglutinin.
Klimpel et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10277-10281 disclose furin-mediated cleavage of anthrax protective antigen.
Molloy et al., 1992, J. Biol. Chem 267: 16396-16402 disclose furin-mediated cleavage of anthrax protective antigen.
Klimpel et al., 1992, Annual Meeting, Amer. Sco. Microbiol. Abst. B-32 disclose furin-mediated cleavage of diphtheria toxin.
Furin can be inhibited by specific peptidyl chloroalkylketones (Garten et al., 1989, Virology 172: 25-31; Molloy et al., 1992 J Biol. Chem., 267:6396-16402), but these substances are toxic in vivo. Given the importance of this endoprotease in activation of bacterial toxins, viral structural proteins and bioactive molecules, there is a need for the development of safe and specific furin inhibitors.